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Culture method for highly expressing erythropoietin in serum-free medium and cho cells

A technology of serum-free medium and culture method, which is applied to the culture field of high-efficiency expression of erythropoietin, can solve the problem of not being able to efficiently and stably express EPO in CHO cells, and is suitable for large-scale production, improves the degree of glycosylation, and has stable expression. Effect

Active Publication Date: 2011-12-07
SHENZHEN SCIPROGEN BIO PHARMA
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

[0003] The purpose of the present invention is to provide a serum-free medium that can be used to improve the expression level and expression stability of EPO for the problem that the current medium cannot meet the high-efficiency and stable expression of EPO in CHO cells, and the serum-free medium can be used in CHO Application of high-efficiency expression of erythropoietin in cells

Method used

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  • Culture method for highly expressing erythropoietin in serum-free medium and cho cells
  • Culture method for highly expressing erythropoietin in serum-free medium and cho cells
  • Culture method for highly expressing erythropoietin in serum-free medium and cho cells

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Embodiment 1

[0028] According to the culture process, the seed cells were activated and amplified, then the cells were digested with trypsin, the cell suspension was prepared, inoculated, and after serum-containing culture was carried out in 10% bovine serum DMEM medium without MTX, the cells were separated in 0mmol / L sodium n-butyrate, 0.5mmol / L sodium n-butyrate, 1.0mmol / L sodium n-butyrate and 2.0mmol / L serum-free medium, the serum-free medium includes a ratio of 1:9 DMEM medium and CHO-S-SFM II medium. After culturing at 36.8±2° C. for 48 hours, the supernatants were collected, and the EPO expression levels in the culture supernatants were measured respectively. The results showed that 1-2mmol / L sodium n-butyrate can not only stabilize the expression of EPO per unit cell, but also have no obvious effect on the growth of cells. see results figure 2 .

Embodiment 2

[0030] According to the culture process, the seed cells were activated and amplified, then the cells were digested with trypsin, the cell suspension was prepared, and inoculated, the cells were added to 10% bovine serum DMEM with 0 μmol / L, 0.5 μmol / L and 1 μmol / L MTX respectively. After serum-containing culture was carried out in the culture medium, serum-free culture was carried out by adding 1.0mmol / L sodium n-butyrate to serum-free medium respectively, and the serum-free medium included DMEM medium and CHO-S-SFM II medium. After culturing at 36.8±2°C for 48 hours, the supernatants were collected, and the expression levels of EPO in the culture supernatants were measured respectively. The results showed that the addition of 0.5-1 μmol / L MTX to the medium could not only stabilize the expression of EPO per unit cell, but also had no obvious effect on the growth of the cells. see results image 3 .

Embodiment 3

[0032] According to the culture process, the seed cells were activated and amplified, then the cells were digested with trypsin, the cell suspension was prepared, inoculated, and after serum-containing culture in 10% bovine serum DMEM medium with 1 μmol / L MTX, the cells were separately Cultivate in serum-free medium without glycosylation reagent and glycosylation reagent containing D-galactose 250mg / L, D-mannose 250mg / L, N-acetylglucosamine 250mg / L, said serum-free The medium includes DMEM medium and CHO-S-SFM II medium with a ratio of 1:9. After culturing at 36.8±2° C. for 48 hours, the supernatants were collected, and the EPO expression levels in the culture supernatants were measured respectively. The results showed that glycosylation reagents could increase the proportion of sialic acid EPO in the culture supernatant. See the results of IEF+Western-blot Figure 4 .

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Abstract

The invention discloses a serum free medium for expressing erythropoietin in CHO (Chinese hamster ovary) cells, and a culture method for high expression of erythropoietin in CHO cells. The serum free medium contains additives D-glucose and sodium butyrate, and also contains one or more of D-galactose, D-mannitose and N-acetylglucosamine. The culture method comprises the steps of: performing serumculture on activated seed cells by using a serum medium, and then performing serum free culture by the serum free medium. By adopting the medium and culture method disclosed by the invention, recombinant human erythropoietin protein of high and stable expression can be obtained, and the glycosylation degree of erythropoietin is improved, i.e. the specific gravity of EPO (erythropoietin) sialic acid is improved. As high as 1.0*107IU recombinant human erythropoietin can be obtained from one liter of culture fluid by the culture method provided by the invention, the expression is stable, and theculture method is simple and suitable for large-scale production.

Description

technical field [0001] The invention relates to the field of animal cell culture, in particular to a serum-free medium for CHO cells expressing erythropoietin and a culture method for highly expressing erythropoin in CHO cells. Background technique [0002] Erythropoietin (Erythropoietin, EPO) is a glycoprotein hormone that stimulates bone marrow hematopoiesis and was first discovered in 1906. It is mainly derived from the kidney (a small amount from the liver) and is synthesized by the interstitial cells surrounding the cortical tubes. Before the birth of gene recombination technology, EPO was mainly extracted from the urine and sheep blood of anemia patients, the yield was very low, and it was extremely unstable. It was difficult to determine its physical, chemical and biological properties, and it could not be applied on a large scale. The gene of erythropoietin was constructed into Chinese hamster ovary cells (CHO cells) by DNA recombination technology to form CHO cells...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12P21/02C12R1/91
Inventor 吴园园盛光阳张涤平吴建祥张自强曲和之黄俊龙刘乐连
Owner SHENZHEN SCIPROGEN BIO PHARMA
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